Over the past several years, we have been studying two phenomena in cloned populations of CD4 positive T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Costimulation entails a 30 to 100-fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 20-50- fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo.During the past year we have made progress in three areas: 1) Last year we demonstrated that the CREB-1/CREM transcription factor showed an increased binding to the minus 180 region of the IL-2 enhancer in anergized T cell clones in their resting state. To determine the functional relevance of this observation we made selective mutations in the sequence around the minus 180 site and hooked them up to a Luciferase reporter gene in a transient transfection assay. We were able to show that a mutation that disrupted the binding of the CREB/CREM complex prevented the inhibition of transcription normally seen in anergy, while a mutation that disrupted the binding of a nearby JUN/OCT complex did not. This result suggests that CREB/CREM complexes may play a key role in the negative regulation of IL-2 transcription. 2) Cycloheximide, a translational inhibitor, has been reported to superinduce IL-2 mRNA partly by stabilizing the IL-2 message. We have now shown that this is not due to inhibition of translation of a labile RNase, because puromycin another translational inhibitor did not have a similar effect. Furthermore, superinduction was not observed in the presence of CD28 costimulation suggesting that cycloheximide may operate by mimicking the CD28 signaling pathway. 3) We have set up a new in vivo model for tolerance to low dose antigens by injecting CD4 positive cytochrome c-specific T cells from T cell receptor transgenic mice on a Rag2-/- background into a second transgenic mouse expressing very low levels of the cytochrome c antigen under the control of the MHC class I promoter and the immunoglobin enhancer. The T cells are initially activated by the antigen and proliferate for several days, but then they reach a steady state level in which their IL-2 response to high doses of antigen in vitro is inhibited 90 percent. The T cells can make small amounts of IFN gamma and IL-10 on restimulation, but not IL-4. The biochemical nature of this state is currently under investigation. - Costimulation, Anergy, Transgenics, Mice, Cytokines, T cells, Transcription, Autoimmunity, Chronic infection, Transfection